Convergence of Wnt Signaling on the HNF4α-Driven Transcription in Controlling Liver Zonation

doi:10.1053/j.gastro.2009.05.038

Gastroenterology

Volume 137, Issue 2, August 2009, Pages 660-672

https://doi.org/10.1053/j.gastro.2009.05.038 Get rights and content

Background & Aims

In each hepatocyte, the specific repertoire of gene expression is influenced by its exact location along the portocentrovenular axis of the hepatic lobule and provides a reason for the liver functions compartmentalization defined “metabolic zonation.” So far, few molecular players controlling genetic programs of periportal (PP) and perivenular (PV) hepatocytes have been identified; the elucidation of zonation mechanisms remains a challenge for experimental hepatology. Recently, a key role in induction and maintenance of the hepatocyte heterogeneity has been ascribed to Wnt/β-catenin pathway. We sought to clarify how this wide-ranging stimulus integrates with hepatocyte specificity.

Methods

Reverse transcriptase polymerase chain reaction (RT-PCR) allowed the transcriptional profiling of hepatocytes derived from in vitro differentiation of liver stem cells. The GSK3β inhibitor 6-bromoindirubin-3′-oxime (BIO) was used for β-catenin stabilization. Co-immunoprecipitations were used to study biochemical protein interactions while ChIP assays allowed the in vivo inspection of PV and PP genes regulatory regions.

Results

We found that spontaneous differentiation of liver stem cells gives rise to PP hepatocytes that, after Wnt pathway activation, switch into PV hepatocytes. Next, we showed that the Wnt downstream player LEF1 interacts with the liver-enriched transcriptional factor HNF4α. Finally, we unveiled that the BIO induced activation of PV genes correlates with LEF1 binding to both its own and HNF4α consensus, and the repression of PP genes correlates with HNF4α displacement from its own consensus.

Conclusion

Our data show a direct and hitherto unknown convergence of the canonical Wnt signaling on the HNF4α-driven transcription providing evidences of a mechanism controlling liver zonated gene expression.

Section snippets

Cell Lines and Culture Conditions

Nontumorigenic, stable RLSC lines, obtained from murine wild-type liver explants at various stages of development, were previously described.¹¹ These cells were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 50 ng/mL epidermal growth factor, 30 ng/mL insulin-like growth factor-II (PeproTech Inc, Rocky Hill, NJ), 10 μg/mL insulin (Roche, Mannheim, Germany), 2 mmol/L l-glutamine, 100 μg/mL penicillin and 100 μg/mL streptomycin (Gibco, Grand Island, NY), using collagen I

Hepatocytes Derived From RLSCs Show a PP Phenotype

We first analyzed the RLSC spontaneous differentiation program. Within a few weeks of culture, these cells, characterized by a flattened morphology and growing in a scattered fashion, spontaneously acquired a cuboidal and tightly packed hepatocyte-like morphology. These differentiated cells, here indicated as RLSC-derived hepatocytes (RLSCdH), expressed the polarity markers zonula occludens 1 and E-cad as well as the liver enriched transcriptional factor HNF4α (Figure 1A).

Next, we analyzed

Discussion

The specific gene expression repertoire of single hepatocyte, is correlated with its anatomic location in the hepatic lobule and, therefore, with the different demand of liver functions. How the liver-specific gene expression is zonally controlled by the microenvironmental signals is a relevant issue in experimental hepatology.

In vivo evidence arising from genetic manipulation and from liver tumors, although indicating the involvement of specific morphogenetic factors (ie, oxygen, Wnts, and

Acknowledgments

M.C. and C.C. contributed equally to this work.

References (26)

R. Gebhardt
Metabolic zonation of the liver: regulation and implications for liver function
Pharmacol Ther
(1992)
S. Benhamouche et al.
Apc tumor suppressor gene is the ”zonation-keeper” of mouse liver
Dev Cell
(2006)
K.M. Cadigan
Wnt-beta-catenin signaling
Curr Biol
(2008)
R.H. Giles et al.
Caught up in a Wnt storm: Wnt signaling in cancer
Biochim Biophys Acta
(2003)
R. Gebhardt et al.
Different proliferative responses of periportal and perivenous hepatocytes to EGF
Biochem Biophys Res Commun
(1991)
A. Nakashima et al.
Cross-talk between Wnt and bone morphogenetic protein 2 (BMP-2) signaling in differentiation pathway of C2C12 myoblasts
J Biol Chem
(2005)
K. Boras et al.
Alx4 binding to LEF-1 regulates N-CAM promoter activity
J Biol Chem
(2002)
A. Kinugasa et al.
Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule
Biochem J
(1986)
R. Gebhardt et al.
Changes in distribution and activity of glutamine synthetase in carbon tetrachloride-induced cirrhosis in the rat: potential role in hyperammonemia
Hepatology
(1994)
Z.D. Burke et al.
The Wnt/beta-catenin pathway: master regulator of liver zonation?
Bioessays
(2006)

A. de La Coste et al.

Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas

Proc Natl Acad Sci U S A

(1998)

M.D. Thompson et al.

WNT/beta-catenin signaling in liver health and disease

Hepatology

(2007)

V.S. Stanulovic et al.

Hepatic HNF4alpha deficiency induces periportal expression of glutamine synthetase and other pericentral enzymes

Hepatology

(2007)

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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC); Ministero della Salute, R.F.; Ministero Università e Ricerca Scientifica (MIUR).

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Basic—Liver, Pancreas, and Biliary TractConvergence of Wnt Signaling on the HNF4α-Driven Transcription in Controlling Liver Zonation

Background & Aims

Methods

Results

Conclusion

Section snippets

Cell Lines and Culture Conditions

Hepatocytes Derived From RLSCs Show a PP Phenotype

Discussion

Acknowledgments

Pharmacol Ther

Dev Cell

Curr Biol

Biochim Biophys Acta

Biochem Biophys Res Commun

J Biol Chem

J Biol Chem

Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule

Biochem J

Changes in distribution and activity of glutamine synthetase in carbon tetrachloride-induced cirrhosis in the rat: potential role in hyperammonemia

Hepatology

The Wnt/beta-catenin pathway: master regulator of liver zonation?

Bioessays

Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas

Proc Natl Acad Sci U S A

WNT/beta-catenin signaling in liver health and disease

Hepatology

Hepatic HNF4alpha deficiency induces periportal expression of glutamine synthetase and other pericentral enzymes

Hepatology

Basic—Liver, Pancreas, and Biliary Tract
Convergence of Wnt Signaling on the HNF4α-Driven Transcription in Controlling Liver Zonation